Mechanism of Phototoxicity of Protoporphyrin and Uroporphyrin on Bovine Corneal Endothelium

Abstract

Porphyrins are potent photosensitizers. This paper reports the mechanisms of phototoxicity produced by protoporphyrin (PP) and uroporphyrin (UP) in corneal endothelium. Corneas from fresh bovine eyes were exposed to ultraviolet- (UV) visible radiation in the presence of PP or UP. The control corneas exposed were either irradiated in the absence of porphyrins or incubated in the dark in the presence of PP or UP. After incubation in the dark or irradiation of the corneas, damage to the endothelium was assessed by morphologic examination after staining with trypan blue and alizarin red S, increase in corneal thickness measured using ultrasound pachymetry, and by determining the quantity of lactate dehydrogenase (LDH) released. The results presented in this paper demonstrate that UV-visible irradiation of bovine corneas in the presence of PP and UP induces endothelial damage. Larger numbers of dead endothelial cells per unit area and larger areas of damage were observed in the corneas irradiated with PP or UP. However the concentration of PP necessary to induce phototoxicity (2–5 μM) was lower than that of UP (5–10 μM). Catalase inhibited the phototoxic effect of UP more than that of PP at the same concentrations. Morphometric analyses of experimental and control corneal endothelium supported our qualitative observation with the vital staining of the corneas. Pachymetric studies show that the corneal thickness increases after irradiation in the presence of PP or UP. However, in contrast to the endothelial damage observed by vital staining and morphometry, the increase in corneal thickness produced by PP or UP was of the same order. Corneal thickness was increased after irradiation in the presence of different concentrations of PP or UP. As the concentration of PP or UP increased, the corneal thickness was proportionately increased. At 5 μM PP or UP the thickness was increased by 27.9% and 37.1%, respectively. Incubation of corneas in the dark with the 5 μM of PP or UP increased the corneal thickness only by 12.3% and 11.8%, respectively. The LDH leakage into the media increased as the concentration of the porphyrin was increased. Addition of catalase to the medium did not affect the LDH release when 1 or 2 μM PP was added. It decreased the LDH release when the PP concentration was 5μM. However, the LDH release at 5 or 10 μM UP was completely abolished by catalase. Therefore, it seems that the UP phototoxicity in cornea is largely due to H₂O₂, whereas the PP phototoxicity is more due to ¹O₂.